Field of the Invention
The present disclosure relates generally to methods for detecting specific molecules in cells, and more specifically, to the use of imagery in methods for quantitating the movement of molecules within a cell, including adherent and non-adherent cells.
Description of the Related Art
Signal transduction pathways regulate most cellular biological processes and have a critical influence on cellular responses to external stimuli. Typically, cell surface receptors that bind to a specific extracellular mediator trigger a cascade of intracellular signaling events that alter cellular metabolism or gene expression, and such changes contribute to the cellular response. The intracellular signaling cascade often involves the translocation of transcription factors or second messengers from the cytoplasm to the nucleus.
Historically, nuclear translocation events have been studied microscopically by observing the sub-cellular localization of fluorescent probe-labeled signaling molecules. Until recently, microscopic applications have been limited due to the subjective nature and the lack of means to quantitate imagery. Currently, several quantitative plate based microscopy platforms are available that attempt to quantitate translocation (ArrayScan, Cellomics, Inc. (Pittsburgh, Pa.); Laser Scanning Cytometer, Compucyte Corporation (Cambridge, Mass.); IN Cell Analyzer, Amersham International plc. (Little Chalfont, England)). However, these microscopy platforms typically rely on the use of adherent cell lines, and their biological responses may differ from suspension-type cells (which include most blood cells).
Traditionally, the measurement of the translocation of fluorescently bound molecules into the nucleus has been determined by a method referred to as the Nuc-Cyt difference (Ding et al., J. Biol. Chem. 273:28897, 1998). This measurement involves the following steps: (1) determining the boundaries of the nucleus which has been stained with a nuclear stain; (2) eroding the mask or area contained within the boundaries to insure the entire area is within the nucleus; (3) summing up the total fluorescence intensity associated from the labeled molecules of interest (Total Nuclear Fluorescence); (4) dilating the nuclear boundary to determine an annular ring solely contained in the cytoplasm and integrate the fluorescence associated with the labeled molecule of interest (Annular Cytoplasm Fluorescence); and (5) calculating the difference between the Total Nuclear and Annular Cytoplasm Fluorescence to yield the Nuc-Cyt difference. However, this method is unlikely to produce the best measurement because it relies on an accurate nuclear mask, subjective erosion and dilation routines that determine the nuclear' and cytoplasmic boundaries, an additional subjective dilation of the cytoplasm mask to create an annular volume, and both the cytoplasm and the nucleus have areas that are not represented in the calculation.
Thus, the need exists for techniques that can allow quantitation of molecular Transport, such as nuclear translocation, in cells in flow to afford the opportunity to study suspension-based cell lines as well as primary cells. For example, such techniques would allow detailed analysis of nuclear translocation responses in subset of cells, such as blood cells. The present invention meets such needs, and further provides other related advantage.